1. Field of the Invention
The present invention relates to novel cyclosporins, processes for their production, their use as pharmaceuticals and pharmaceutical compositions comprising them. Furthermore, this invention discloses a novel general method for the exchange of substituents at the sarcosine residue of the cyclosporin macrocycle.
2. Description of the Related Art
Cyclosporin A is well known for its immunosuppressive and antiinflammatory properties but many biological properties have been described in addition. EP 0 194 972 describes cyclosporin derivatives with substituents on the sarcosine in position 3 of the macrocycle, the introduction of such substituents, as well as the immunosuppressive, antiinflammatory and antiparasitic activity of these cyclosporin derivatives. EP 0 484 281 describes cyclosporin derivatives with reduced immuno-suppressive potency and activity against HIV.
The present invention discloses novel cyclosporins which can be used for the treatment of infectious diseases, of chronic inflammatory and autoimmune diseases, to prevent cardiac hypertrophy, to treat and prevent ischemia and reperfusion injury, to treat neurodegenerative diseases, and to induce processes of tissue regeneration.
A second embodiment of the present invention is a novel method to prepare cyclosporins with substituents at the sarcosine in position 3 of the macrocycle. EP 0 194 972 describes the introduction of certain substituents at the sarcosine. The method described in EP 0 194 972 involves treatment of a cyclosporin with strong base to generate a polyanion and subsequent reaction of this polyanion with electrophiles, such as disulfides, alkyl halides or other suitable alkylating agents. Halogens or sources of positive halogen can also be used, as well as aldehydes. There is no example in the prior art which describes the exchange of such a substituent by another. The present invention discloses such a method. In this novel method, a suitable substituent is first introduced into a cyclosporin polyanion and the resulting product is isolated. The substituent is subsequently activated to become a leaving group and replaced by the desired novel substituent. This novel method allows the introduction of a wide variety of substituents into the sarcosine residue of the cyclosporin macrocycle.
The cyclosporin nomenclature and numbering systems used hereafter are those used by J. Kallen et al., xe2x80x9cCyclosporins: Recent Developments in Biosynthesis, Pharmacology and Biology, and Clinical Applicationsxe2x80x9d, Biotechnology, second edition, H.-J. Rehm and G. Reed, ed., 1997, p535-591 and are shown below:
Objects of the present invention are therefore compounds of the formula I 
and their pharmaceutically acceptable salts wherein the letters A to L represent residues of the following amino acids:
A (L)-alpha-N-methylamino-beta-hydroxy acid of the formula II, 
xe2x80x83wherein R1 is (E)-2-butenyl-1,
B alpha-amino-butyric acid, alpha-amino-valerianic acid (norvaline), threonine, or valine,
C substituted sarcosine of the formula III 
xe2x80x83in which x is
Sxe2x80x94(O)nxe2x80x94R2, in which n has the value zero, one or two, and R2 is hydrogen, unsubstituted or substituted, unbranched or branched, acyclic, monocyclic or polycyclic, saturated or unsaturated lower alkyl, substituted or unsubstituted aryl or heteroaryl, or
X is Oxe2x80x94R3, in which R3 is hydrogen, unsubstituted or substituted, unbranched or branched, saturated or unsaturated, acyclic, monocyclic or polycyclic lower alkyl, or acyl, or
X is sulfonium groups of the formula IV, 
xe2x80x83in which R4 and R5 are independently selected from lower alkyl, aryl, or heteroaryl and Yxe2x88x92 is an anion, or
X is a group of the formula V, 
xe2x80x83in which R6 and R7 are independently selected from lower alkyl or aryl or form together a ring and R8 is hydrogen or substituted or unsubstituted lower alkyl and Yxe2x88x92 is an anion, or
C is a residue of the formula VI and Yxe2x88x92 is an anion,
[Hxe2x80x94N+(CH3)xe2x95x90CHxe2x80x94COxe2x80x94OH]Yxe2x88x92xe2x80x83xe2x80x83Formula VI
D N-methyl-leucine, gamma-hydroxy-N-methyl-leucine, N-methyl-valine, or N-methyl-isoleucine,
E valine,
F N-methyl-leucine,
G alanine,
H glycine, (D)-alanine, (D)-serine, O-hydroxyethyl-(D)-serine,
I,K N-methyl-leucine, and
L N-methyl-valine.
The (E)-2-butenyl-1 rest in A has preferrably the trans configuration.
Examples for lower alkyl are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, isopentyl, tert-pentyl, neopentyl, hexyl and its isomers.
Examples for monocyclic lower alkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
Examples for polycyclic lower alkyl groups are bicylo[2.1.1]hexyl, norbornyl, bicyclo[2.2.2]octyl.
Examples for unsaturated lower alkyl are vinyl, allyl, butenyl, pentenyl, pentadienyl, hexenyl, hexadienyl.
Examples for substituents in these radicals are hydroxy, methoxy, ethoxy, propoxy, isopropoxy, amino, monoalkylamino, dialkylamino, acylamino, halogen, acyl, carboxy, carbamido.
Examples for monoalkylamino are methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino, tert-butylamino, 1-pentylamino, 2-pentylamino, 3-pentylamino, isopentylamino, tert-pentylamino, neopentylamino, hexylamino and its isomers.
Examples for dialkylamino are N,N-dimethylamino, N-methyl-N-ethylamino, N,N-diethylamino, N-propyl-N-methylamino, N-methyl-N-isopropylamino, dipropylamino, diisopropylamino, N-butyl-N-methylamino and its isomers, N-butyl-N-ethylamino and its isomers, N-butyl-N-propylamino and its isomers, N,N-dibutylamino and its isomers.
The alkyl groups of dialkylamino may also form a ring together. Examples are azetidine, pyrrolidine, piperidine, morpholine, piperazine, Nxe2x80x2-alkylpiperazine, azabicylo[2.1.1]hexane, azanorbornane, azabicyclo[2.2.2]octane.
Examples for acyl are formyl, acetyl, propionyl, butyryl, pivaloyl, benzoyl, alkoxycarbonyl.
Examples for acylamino are N-formylamino, N-acetylamino, N-tert-butoxycarbonyl-amino, N-benzyloxycarbonyl-amino, N-benzoyl-amino, N-phthaloyl.
Examples for substituted aryl are tolyl, chlorphenyl, methoxyphenyl, aminophenyl, dimethylaminophenyl, 1-naphthyl, 2-naphthyl.
Examples for heteroaryl are thiazole, oxazole, imidazole, pyridine, pyrazole, pyrimidine, pyrazine, triazine, benzthiazole, benzoxazole, benzimidazole.
Examples of sulfonium groups are dimethylsulfonium, S-methyl-S-phenylsulfonium, S-methyl-S-allylsulfonium, S-methyl-S-carboxamidomethyl-sulfonium, S-dodecyl-S-methylsulfonium.
Examples for groups of the formula V are N-methyl-pyridinium-2-ylthio, N-methyl-pyridinium-4-ylthio, N-methyl-triazinylthio, N-methyl-benzthiazol-2-ylthio, pyridinium-2-ylthio toluenesulfonate, pyridinium-4-ylthio toluenesulfonate, pyridinium-2-ylthio methanesulfonate.
Compounds of the formula I in which C is a sarcosine substituted by Sxe2x80x94R2 are prepared by forming polyanions from cyclosporins in which C is sarcosine and reacting these polyanions with appropriate sulfur electrophiles like disulfides, thiolsulfinates, sulfenyl halides, or disulfide-derived sulfonium salts. The polyanions are in turn prepared by treating the cyclosporins in an appropriate solvent at low temperature with an excess of a strong base. Examples for strong bases are alkali amides like lithium amide, natrium amide, lithium diisopropylamide or lithium hexamethyl disilazide. Examples for inert solvents used for these reactions are tetrahydrofurane, dioxane, diethylether, methyl tert-butylether, or liquid ammonia.
Compounds of the formula I in which C is sarcosine substituted by Sxe2x80x94R2 are furthermore prepared by exchanging the substituent X in compounds of formula I in which C is sarcosine substituted by X and where X is a residue of formula IV or formula V by thiols HSxe2x80x94R2, in which R2 is lower alkyl, aryl, or acyl.
A compound of the formula I in which C is sarcosine substituted by Sxe2x80x94R2 in which R2 is hydrogen is prepared by treating compounds of formula I in which C is sarcosine substituted by Sxe2x80x94R2 and in which R2 is acyl in an appropriate solvent with ammonia, hydrazine, hydroxylamine, or organic derivatives thereof, such as methylamine, benzylamine, methylhydrazine, or dimethylhydrazine. Appropriate solvents for these reactions are alcohols, such as methanol or ethanol, ethers such as diethylether, tetrahydrofurane, or dioxane, or inert aprotic solvents, such as dimethyl formamide. The resulting thiol can be isolated but is more conveniently alkylated directly by adding to these reactions an alkylating agent such as an alkyl halide or esters of alcohols with sulfuric acid, or organic sulfonic acids such as 4-toluene sulfonic acid, methane sulfonic acid, or trifluoromethane sulfonic acid. The present invention makes it therefore possible to produce compounds of the formula I in which C is sarcosine substituted by Sxe2x80x94R2 in which R2 is substituted or unsubstituted, branched or unbranched, saturated or unsaturated, acyclic, monocyclic, or polycyclic lower alkyl.
Compounds of the formula I in which C is a sarcosine substituted by SOxe2x80x94R2 and by SO2xe2x80x94R2 are prepared by treating compounds of formula I in which C is sarcosine substituted by Sxe2x80x94R2 with an appropriate oxidant in an inert solvent. Examples for such oxidants are hydrogen peroxide, sodium chlorate, sodium periodate, peroxyacetic acid, meta-chloroperbenzoic acid, or potassium persulfate. Solvents for these reactions are for example mixtures of water with organic solvents such as tetrahydrofurane, dioxane or acetic acid, or anhydrous organic solvents such as dichloromethane, chloroform, tetrachloroethane, tetrahydrofurane, or dioxane.
Compounds of the formula I in which C is a sarcosine substituted by Oxe2x80x94R3 can be prepared by exchanging the substituent Sxe2x80x94R2 in compounds of formula I in which C is sarcosine substituted by Sxe2x80x94R2. This exchange reaction is effected by metal salts which have an affinity for sulfur, such as mercuric acetate, silver acetate, copper acetate and others, but can also be effected by the presence of Bronsted or Lewis acids. The acidic nature of Bronsted acids is due to their capacity to act as proton donors. Such acids are for example sulfuric acid, toluene sulfonic or camphersulfonic acid, hydrochloric acid, but also acetic acid, formic acid and other organic carboxylic acids. Lewis acids are compounds having affinity for free electron pairs and forming coordination complexes with groups having free electron pairs. Examples for Lewis acids are boron trifluoride, titanium tetrachloride, aluminum chloride, or zinc chloride. Such Bronsted or Lewis acids or metal salts convert Sxe2x80x94R2 or Oxe2x80x94Rxe2x80x23 substituents at the sarcosine position of the cyclosporin macrocycle into leaving groups, forming an intermediary cation of the formula VI which can then further react with nucleophiles present in the reaction mixture to form the desired products.
The present invention makes it therefore also possible to exchange one given Oxe2x80x94R3 substituent for another Oxe2x80x94R3xe2x80x2 substituent by using the reaction conditions as described above.
A compound of formula I in which the amino acid residue of C is the cation of Formula VII is a common intermediate for these exchange reactions. This type of cation is well known to experts in the field and is analogous to the commonly accepted intermediate in the Mannich reaction. In the case of cyclosporins, however, such an intermediate has never been described and is new. Mannich reactions are used to introduce aminoalkyl residues into a wide variety of nucleophiles, such as enols, phenols, enamines, heterocycles such as indole, pyrrol, or furane. Other nucleophiles reacting with such cations are allyl and vinylsilanes and -stannanes as well as acetylenes. Therefore, cyclosporins in which the amino acid residue of C is the cation of Formula VII are an especially preferred embodiment of the present invention.
The compounds of the present invention act on enzymes called cyclophilins and inhibit their catalytic activity. Cyclophilins occur in a wide variety of different organisms, including human, yeast, bacteria, protozoa, metazoa, insects, plants, or viruses. In the case of infectious organisms, inhibition of the cyclophilin catalytic activity by compounds of the present invention often results in an inhibitory effect on the organism. Furthermore, in humans the catalytic activity of cyclophilins plays a role in many different disease situations. Inhibition of this catalytic activity is often associated to a therapeutic effect. Therefore, the compounds of the present invention can be used for the treatment of infections including that by HIV as well as fungal pathogens, protozoan and metazoan parasites. Furthermore, the compounds of the present invention can be used to treat chronic inflammatory and autoimmune diseases including but not limited to rheumatoid arthritis, psoriasis, and uveitis. In addition, the compounds of the present invention can be used to treat neurodegenerative diseases such as Alzheimer""s disease, Parkinson""s disease, and neuropathies.
Another use of the compounds of the present invention is protection against tissue damage associated to ischemia and reperfusion such as paralytic damage after spinal cord or head injuries or cardiac damage after myocardial infarct.
Furthermore, the compounds of the present invention induce regenerative processes such as that of hair, liver, gingiva, or nerve tissue damaged or lost due to injury or other underlying pathologies, such as damage of the optical nerve in glaucoma.
The compounds of the present invention can, together with pharmaceutically acceptable additives and/or excipients be administered either orally in the form of capsules, tablets, or drink solutions or parenterally in form of acute injections or infusions. They can also be applied locally in form of solutions, eye drops, or as gels and ointments. For topical and parenteral applications it is of special advantage that, unlike cyclosporin A, many of the compounds of the present invention have basic substituents which enable the formation of salts with physiologically acceptable acids.
The daily administered dosage depends on the structure of the medicament, the disease to be treated, and the type of formulation and is from about 1 mg to about 200 mg per kg body weight.